Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

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Chapter 14

EXTRACELLULAR PROTEINASES

IN OCULAR ANGIOGENESIS

Arup Das and Paul G. McGuire

Division of Ophthalmology, Department of Surgery, and Department of Cell Biology & Physiology, University of New Mexico School of Medicine, and New Mexico VA Health Care System, Albuquerque, New Mexico

1.INTRODUCTION

The angiogenesis cascade consists of several phases; upregulation of angiogenic growth factors is followed by increased expression of specific integrins and extracellular proteinases. The invasive process of cell migration through the basement membrane and extracellular matrix (ECM) is facilitated by these proteinases. The phenotype of endothelial cells activated during the proliferative and invasive phases of angiogenesis includes increased expression of cell-substrate adhesion molecules and proteolytic enzymes. Their action facilitates the degradation of the capillary

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basement membrane and migration and subsequent invasion of activated endothelial cells into the surrounding tissues.1-4

The enzymes primarily involved in this process are the serine proteinase, urokinase plasminogen activator (uPA), and members of the matrix metalloproteinase (MMP) family. Upregulation of proteinases is a crucial event in tumor as well as ocular angiogenesis. Pharmacological intervention in this pathway has proved to be an alternative therapeutic approach in preclinical angiogenesis studies, and some of these drugs are now in various phases of clinical trials.

2.UROKINASE (uPA-uPAR SYSTEM)

The proteolytically active urokinase on the cell surface is critical for cell migration. Urokinase is secreted as a single chain proenzyme that can be cleaved by plasmin. Urokinase is present in two molecular forms: a 54 kDa high molecular weight form and a 33 kDa low molecular weight form, which lacks the amino-terminal fragment (ATF) of the protein.5-7 The ATF plays a role in cell proliferation.8,9 The main function of the urokinase is to convert the inactive zymogen form of the enzyme plasminogen to plasmin, a broadspectrum proteinase, which can cleave a variety of ECM components including collagen IV, fibronectin, and elastin. The uPA localizes to the surface of endothelial cells by binding to the uPA receptor (uPAR). This interaction of uPA and uPAR facilitates cell migration through localized proteolytic and nonproteolytic regulation of cell-substrate adhesion.10-11 Recent studies emphasize that the uPAR plays the role of a “versatile orchestrator” and that uPAR, integrin, and very-low-density lipoprotein receptor (VLDLR) interact with each other, resulting in the cycled attachment, detachment, and reattachment of integrins that is necessary for cell migration.12 uPA also activates several MMPs, causing the release of growth factors.10 The uPA-uPAR system has also been implicated in the regulation of cell migration and matrix remodeling involved in angiogenesis both in normal development and in tumor progression and metastasis.10-12

3.MATRIX METALLOPROTEINASES

The MMPs are a family of enzymes involved in the degradation of a variety of ECM components including the collagens, laminin, fibronectin, elastin, and the core protein of proteoglycans.13 Currently, at least 21 members of the MMP family have been identified.13 All the MMPs contain a zinc ion at the active site and show consistent structural and sequence homologies. All are

secreted as latent pro-enzymes and are activated by partial proteolytic cleavage. The MMPs are divided into five subclasses based upon their substrate specificity: the interstitial collagenases (MMP-1, MMP-8 and MMP-13), the gelatinases (MMP-2 and MMP-9), the stromelysins (MMP-3, MMP-10, and MMP-11), the other MMPs (matrilysin or MMP-7; metalloelastase or MMP-12) and the membrane type MMPs (MT-MMP).14 These proteinases play important roles in a variety of cellular process including the regulation of cell migration, proliferation, and apoptosis. MMPs are involved in the normal physiological processes of embryonic development and wound healing and are overexpressed in diseases such as cancer and arthritis. MMPs are upregulated in most types of human cancer and are often associated with a poor prognosis. Some of the MMPs are expressed by tumor cells, while other MMPs are expressed by stromal cells, including endothelial cells, fibroblasts, myofibroblasts, and inflammatory cells.15 The roles of both uPA and MMPs in cell migration in angiogenesis are summarized in Figure 1.

Figure 14-1. Flowchart describing the role of extracellular proteinases in angiogenesis. The urokinase (uPA) acts on the receptor, uPAR, and the activated uPA then converts plasminogen to plasmin, which can degrade the extracellular matrix (ECM) components, as well as activate the MMPs. The MMPs have several functions, including degradation of ECM, breakdown of cell-matrix adhesions and cell-cell adhesions, exposure of cryptic sites, release of VEGF from the ECM, and degradation of PEDF. The combined activity of these proteinases ultimately regulates the cell adhesion and migration necessary for angiogenesis.

4.PROTEINASES IN OCULAR ANGIOGENESIS

Do proteinases play any role in ocular angiogenesis? We examined proteinases by zymography (a technique to quantify proteolytic activity) in retinal extracts from animals with retinal neovascularization (NV) on day 17 (the active angiogenic phase). Significant increases in the high (54 kDa) and low (32 kDa) molecular weight forms of uPA were observed in the retinas of animals with active NV.16 Similar increases were also found in the levels of the proenzyme and active forms of both MMP-2 (72 kDa and 62 kDa, respectively) and MMP-9 (92 kDa and 84 kDa, respectively) in animals with NV. These results suggest that the active phase of the angiogenic process is associated with the increased expression of uPA, MMP-2, and MMP-9. RTPCR studies in experimental animals with retinal NV also revealed increases in mRNA levels for MMP-2, MMP-9, and MT-MMP that correlated with changes in proteinase levels and proenzyme activation.16 MMP-2 is a substrate for MT-MMP, which may explain the presence of increased levels of the activated form of MMP-2. Analysis of mRNA did not detect expression of MMP-3 or MMP-7 in either control or experimental animals, confirming the results of zymographic analysis.

Exposure of a collagen type IV cryptic epitope (a protein sequence that normally remains hidden) represents one of the earliest remodeling events required before vessel sprouting.17 Exposure of these cryptic sites has been found to be inhibited in MMP-9-deficient but not MMP-2-deficient mice, suggesting a role of MMP-9 in their exposure. This would be a novel mechanism in which MMP-9 facilitates angiogenesis by promoting retinal endothelial cell migration and angiogenesis.

We also reported an upregulation of the urokinase receptor, uPAR, in the retinas of a murine model of retinal NV.18 The uPAR protein was localized to vessel profiles within the superficial portion of the retina and to vessels on the vitreal side of the inner limiting membrane. To determine whether uPAR is necessary for the development of retinal NV, we subjected uPAR knockout mice to the same oxygen protocol as used for the murine model of retinal NV and quantified the extent of NV. Retinal NV in uPAR knockout animals was reduced by 73% compared to normal mice,18 and these knockout mice showed normal retinal vascular development. Thus, increased expression of proteinases was observed in the retinas of an animal model with retinal NV, indicating an activation of the proteolytic cascade during angiogenesis.

These animal results were found to correlate with results from a study of proteinases in epiretinal neovascular membranes removed surgically from patients with proliferative diabetic retinopathy.19 The levels of uPA, MMP-2,

and MMP-9 were significantly elevated in the neovascular membranes compared to normal retinas.

Does the upregulation of proteinases also happen in choroidal neovascularization (CNV)? RT-PCR studies have shown upregulation of uPA and uPAR in the choroidal tissues of mice with laser-induced CNV as

well as in CNV membranes from patients with age-related macular degeneration.20,21 We also found that uPAR localized to the endothelial cells

of the fibrovascular tissue within the CNV complex in the laser-induced NV model. Studies with single-gene-deficient mice have shown that the absence of uPA, tPA (tissue plasminogen activator), or plasminogen significantly decreased the development of experimental CNV and that this effect could be explained by a modulation of MMP activity in the laser-induced wounds.20

If proteinases have a role in angiogenesis, are they also involved in early diabetic retinopathy? One of the early features of diabetic retinopathy is the alteration of the blood-retina barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. We investigated the role of extracellular proteinases during the early stages of diabetic retinopathy, especially in relation to the BRB. We have shown in streptozotocin (STZ) treated diabetic Sprague Dawley rats a 1.7-fold increase in retinal vascular permeability after 12 weeks of diabetes and upregulation of the levels of specific extracellular proteinases in the retina compared to non-diabetic controls.22 Using conventional semi-quantitative RT-PCR, MMP-2, MMP-9, and MMP-14 mRNA levels were found to be significantly elevated in the retinas of diabetic animals. Real time RT-PCR was utilized to quantify the mRNA levels for components of the urokinase system in diabetic retinas, and all components of this system were found to be significantly elevated in the 12 week diabetic rats when compared to non-diabetic controls. How do the proteinases function in early diabetic retinopathy? Is there any role of MMPs and urokinase in regulating tight junction functions? Retinal endothelial and pigment epithelial cells treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased transepithelial electrical resistance (TER).22 Western blot analysis of cell extracts treated with MMP-2 or MMP-9 revealed specific degradation of the tight junction protein, occludin. A previous study reported that uPA can regulate the paracellular permeability pathway in VEGFtreated cultured endothelial cells.23 Increased vascular permeability in experimental diabetes is associated with reduced endothelial occludin content,24 and VEGF has been shown to cause rapid phosphorylation of occludin.25 Thus, elevated expression of MMPs in the retinas of diabetic animals may facilitate an increased vascular permeability by a mechanism involving proteolytic degradation of the occludin followed by disruption of

the overall tight junction complex. A greater understanding of the role of proteinases in altering tight junction proteins may provide future targets for therapeutic intervention.

5.ENDOGENOUS PROTEINASE INHIBITORS

The balance of proteinases and inhibitors has been shown to be a critical determinant of endothelial cell morphology and tube formation in vitro.

5.1Tissue inhibitors of metalloproteinases

MMP activity is in part regulated by the tissue inhibitors of metalloproteinases (TIMPs), which bind the proteinases and inhibit their activity. Four TIMPs have been identified, and they share significant

homology at the amino acid level, including 12 cysteine residues that form six disulfide bonds in each member of the family.14,26 Each TIMP is capable

of inhibiting all metalloproteinases; however, preferential binding to specific MMPs has been reported.27,28 TIMP-1 primarily inhibits the activities of MMP-1, -3, and -9, whereas TIMP-2 inhibits MMP-2.27,29 TIMP-2 has also

been shown to bind and stabilize MMP-2 by preventing autolytic degradation and by participating in its activation.30,31 TIMP-3 is localized exclusively to the ECM and is relatively insoluble, illustrating its potential to prevent matrix proteolysis and the release of growth factors sequestered in the ECM.27 TIMP-3 is present in Bruch's membrane of normal human eyes,32

and TIMP-3 mRNA has been localized to mouse and human retinal pigment epithelial cells.33,34 A point mutation in the TIMP-3 gene has been implicated

in patients with Sorsby's fundus dystrophy, an autosomal dominant macular disease with earlier onset of symptoms similar to those of age-related macular degeneration and characterized by choroidal NV.35,36 The TIMP-3 content in Bruch's membrane of the macula shows a significant increase in eyes with age-related macular degeneration compared with age-matched normal eyes.37 We have found that the TIMP-2 message and protein levels in retinas of normal mice in room air increased steadily until day 17, whereas in animals with retinal NV, TIMP-2 mRNA and protein remained significantly lower than in control animals.38 We could not find any significant changes in TIMP-1 and TIMP-3 levels in retinas with NV. Thus, at least in an animal model of retinal NV, we have shown a temporal correlation between proteinases (MMP-2, MMP-9, and MT1-MMP) and TIMP-2 in response to hypoxic stimulation.